Estrogens are known in induce tumors in laboratory animals. In humans, the administration of estrogens has been associated with a high incidence of endometrial carcinoma and, more recently, of breast cancer. In this project, we propose to synthesize and test several estrogens modified in a way to be hormonally highly potent yet completely noncarcinogenic. In our work during the past funding period, we demonstrated that hormonal potency of estrogens and carcinogenic activity are not inherently associated with each other but are separable properties of substances. It is expected that steroid estrogens which can not form any catechol metabolites, will lack carcinogenic activity. Such substances are needed to prove that catechol estrogens are the carcinogenic metabolites of estrogens and to provide 'safe' estrogen medication for human use. Specifically, we will synthesize 11beta-methoxy-4-fluoroestradiol, 11beta-methyl-4-fluoroestradiol, and 2- fluoro-6-methylestradiol and test these estrogens in vitro and in vivo for their estrogenic activity, toxicity and effect on metabolizing enzymes. The toxicity in vitro will be assessed by 1. measuring catechol formation by hamster liver and kidney microsomes using a product isolation assay, 2. determining the methylation of catechol metabolites synthesized from modified estrogens, 3. examining the influence of chronic administration of modified estrogens on hamster liver and kidney estrogen 2- and/or 4- hydroxylases, 4. measuring microsome-mediated DNA adduct formation in vitro using 32P-postlabeling analysis, 5. measuring microsome-mediated protein damage by a newly developed NaB3H4 reduction assay. In vivo the estrogenic potency and lack of carcinogenic activity will be tested in Syrian hamsters and in mice. Tissues from these animals will also be investigated for DNA adduct formation and protein damage, and concentrations of estrogen and catechol estrogen (if formed).